The completion of the genome sequencing of humans and other species and the emergence of new technologies in mass spectrometry have together fostered unprecedented opportunities for studying proteins on a large scale. It is expected that large scale quantitative measurements of protein expressions in different sets of samples, referred to as comparative proteomics, will advance our understanding of physiological processes and disease mechanisms. Comparative proteomic approaches have been applied to various biological samples to identify and quantify proteins that are up- or down-regulated in response to biological conditions. To date, there are two primary strategies used in current comparative proteomics; two dimensional gel electrophoresis (2D-PAGE) based strategy and mass spectrometry based in vitro stable isotope labeling strategy.
Although 2D-PAGE based methods have been a primary choice in comparative proteomics, 2D-gels are cumbersome to run, have a poor dynamic range, and are biased toward abundant and soluble proteins. In contrast, the mass spectrometry based stable isotope labeling strategy has a potential of overcoming most of the weaknesses of the 2D-PAGE based methods. If the stable isotope labeling can be achieved efficiently and equivalently for each distinct sample, then two samples are compared using isotopic ratios. Among the in vitro stable isotope labeling methods, proteolytic 18O labeling is the simplest stable isotope labeling method and is expected to have the least methodological error (technical variations). Therefore, the proteolytic 18O labeling method has a potential to be a central method in comparative proteomics.
Although promising, a major drawback of the proteolytic 18O labeling method has been the generation of a mixture of isotopic isoforms upon proteolytic digestion resulting from the differential incorporation of either one or two 18O atoms (18O1/18O2) into each digested peptide species generated. Typical serine proteases used include trypsin, Lys-C or Glu-C proteases. Unfortunately, past studies have found that the ratios of the first and the second 18O atom incorporation vary significantly with peptide sequences, and thus, the ratios of 18O1- and 18O2-peptides cannot be predicted with any certainty. The quantifications of the peptides results in significant errors in calculating 16O- and 18O-labeled peptide ratios. In spite of more recent wide appreciation of this problem, no method has been reported to solve the problem.
A second significant drawback of using serine proteases that has been demonstrated for 18O labeling is that digested peptide products continue to react with these proteases at the carboxyl termini. As a result, the serine proteases will catalyze oxygen back-exchange reaction when two digests, the first in H216O and the second in H218O, are mixed together. A previous report demonstrated that trypsin catalyzed oxygen back-exchange reaction occurs and leads to inaccurate quantification.